WO2019039781A2 - Novel aeromonas salmonicida bacteriophage aer-sap-2 and use thereof in inhibiting proliferation of aeromonas salmonicida bacteria - Google Patents

Abstract

The present invention relates to Myoviridae bacteriophage Aer-SAP-2(accession number KCTC 12909BP), isolated from nature, having an ability to kill Aeromonas salmonicida bacteria, and a genome represented by SEQ ID NO: 1, and a method for preventing or treating a disease caused by Aeromonas salmonicida bacteria, using a composition comprising the same bacteriophage as an effective ingredient.

Description

The novel eromonas salmonidia bacteriophage Aer-SAP-2 and its eromonas salmonidase proliferation inhibiting uses

The present invention relates to a bacteriophage isolated from nature capable of killing eromonas salmonididae by infection with eromonas salmonididae, and a composition containing the same as an effective ingredient to prevent diseases caused by eromonas salmonididae Characterized in that it has the ability to kill eromonas salmonididae and has the genome of SEQ ID NO: 1, characterized in that the mio viridis versus bacteriophage Aer-SAP -2 (Accession No. KCTC 12909BP), and a method for preventing or treating a disease caused by eromonas salmonidia bacteria using the composition comprising the bacteriophage as an active ingredient.

Aeromonas salmonicida , belonging to Aeromonadaceae , is a tuberous anaerobic gram-negative bacterium and is a non-invasive mononuclear bacterium that causes serious diseases such as salmonid and salmonid fish such as salmon and rainbow trout. Is known as a causative organism. Eromonas salmonidida has a homogeneous serotype and is known to have a thermostable, O antigen. Eromonas salmonidida, which has a thermostable bacterial antigen, is pathogenic to sepsis and is known to cause systemic infection especially when it has antigen group O3.

Eromonas salmonidida may cause extensive bacterial septicemia or furunculosis in fish and may cause massive mortality in severe cases and cause significant economic loss in the fish industry . Therefore, it is urgent to develop a method that can be used to prevent the infection of eromonas salmonidida and further to treat the infection.

Recently, various antibiotics have been used for the prevention and treatment of diseases caused by eromonas salmonidida. However, as the incidence of resistant bacteria to these antibiotics increases, it is urgent to secure other measures besides antibiotics.

Recently, the use of bacteriophage as a countermeasure against bacterial infectious diseases has received much attention. Especially, it is receiving more attention because of its excellent antibacterial activity against antibiotic resistant bacteria. Bacteriophage is a very small microorganism that infects bacteria, usually called phage. The bacteriophage has the ability to kill bacterial cells by infecting the bacteria inside the cells after infecting the bacteria and destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria after the proliferation. The bacterium infection method of bacteriophage is highly specific, and the types of bacteriophages that can infect specific bacteria are limited to some. That is, certain bacteriophages can infect only a specific category of bacteria, and thus certain bacteriophages can provide an antibacterial effect only for certain bacteria. Due to the bacterium specificity of these bacteriophages, bacteriophage provides an antimicrobial effect only on the bacteria of interest and does not affect the environment or bacteria in the environment. Conventional antibiotics, which have been widely used for bacterial treatment, have simultaneously influenced several kinds of bacteria. This has caused problems such as environmental contamination and disturbance of normal flora of animals. In contrast, bacteriophages operate only on specific bacteria, so that the use of bacteriophages does not cause a total disturbance in the body. Therefore, the use of bacteriophage is very safe as compared with the use of antibiotics, and the possibility of side effects caused by use is relatively low.

Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer. In 1917, the French bacteriologist d'Herelle discovered that there was an effect of dissolving Shigella dysenteriae in the filtrate of heterozygous patients. By studying this, we found bacteriophage independently and eaten bacteria Called bacteriophage in the sense of. Since then, bacteriophages have been found in many pathogenic bacteria such as dysentery, typhoid, and cholera.

Due to the special ability to kill bacteria, bacteriophage has been expected to be effective as a countermeasure against bacterial infection since its discovery. However, since the discovery of penicillin by Fleming, the spread of antibiotics has become common, and studies of bacteriophage have been limited to some Eastern European countries and the Soviet Union. However, since 2000, the limitations of existing antibiotics have appeared due to the increase of antibiotic resistant bacteria, and bacteriophages have been attracting attention as an anti - bacterial agent due to the possibility of development as a substitute for existing antibiotics.

As described above, bacteriophages are highly specific for bacteria. Because of the high specificity of these bacteriophages to bacteria, bacteriophages often exhibit antibacterial effects only on some strains, even if they belong to the same species (Species). In addition, the intensity of the antibacterial activity of bacteriophages exhibited according to the target bacterial strain may be different. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to obtain an effective control method for a certain kind of bacteria. In order to develop an effective method of using bacteriophage in response to eromonas salmonidida, it is necessary to secure various kinds of bacteriophages that can provide antibacterial effect against eromonas salmonidida, and furthermore, it is necessary to secure various bacteriophages It is necessary to select bacteriophages that have comparative advantages in terms of antimicrobial activity and antibacterial activity.

Accordingly, the present inventors have developed a composition that can be utilized for the prevention or treatment of diseases caused by eromonas salmonididae using bacteriophages isolated from nature capable of killing eromonas salmonididae Further, after trying to develop a method for preventing or treating a disease caused by eromonas salmonididae by using this composition, a bacteriophage suitable for this is separated from nature, and the separated bacteriophage is distinguished from other bacteriophages The present inventors have developed a composition comprising the bacteriophage as an active ingredient after securing the sequence information of the genome so as to be able to construct the erythromycin, To complete the present invention Respectively.

Accordingly, an object of the present invention is to provide a Myoviridae bacteriophage Aer-SAP ( hereinafter referred to as " bacteriophage ") isolated from nature, which has the ability to specifically kill eromonas salmonididae and has a genome represented by SEQ ID NO: -2 (accession number KCTC 12909BP).

It is still another object of the present invention to provide a method for producing eromonas salmonidosis, which contains erythromonas salmonididae as an active ingredient by using a bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) And to provide a composition that can be used for the purpose of preventing or treating a disease caused by Salmonidid bacteria.

It is another object of the present invention to provide a method for producing eromonas salmonidia, which comprises an isolated bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) capable of infecting eromonas salmonididae and capable of killing eromonas salmonidosis, The present invention provides a method for preventing or treating a disease caused by eromonas salmonidosis using a composition that can be used for the purpose of preventing or treating diseases caused by Shido bacteria.

It is still another object of the present invention to provide a bathing agent used for the purpose of preventing or treating diseases caused by eromonas salmonidia bacteria using the above compositions.

It is still another object of the present invention to provide a feed additive for the purpose of providing a specification effect through prevention or treatment of a disease caused by eromonas salmonidosis using the above compositions.

The present invention relates to a microorganism isolated from nature, which has the ability to specifically kill eromonas salmonididae and has a genome represented by SEQ ID NO: 1, and a microorganism isolated from bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP), and a method for preventing or treating a disease caused by eromonas salmonidia bacteria using the composition containing the same as an active ingredient.

Bacteriophage Aer-SAP-2 has been deposited with the Korean Resource Center for Biotechnology (Accession No. KCTC 12909BP) on Sep. 22, 2015, after being separated by the present inventors.

The present invention also provides a bathing agent and a feed additive comprising bacteriophage Aer-SAP-2, which can be utilized for preventing or treating diseases caused by eromonas salmonidia, as an active ingredient.

Since the bacteriophage Aer-SAP-2 contained in the composition of the present invention effectively kills eromonas salmonididae, it is effective for prevention (prevention of infection) and treatment (infection treatment) of diseases caused by eromonas salmonididae . Therefore, the composition of the present invention can be utilized for the purpose of preventing or treating diseases caused by eromonas salmonidia.

As used herein, the terms " prevent " or " prophylaxis " refer to (i) prevention of infection of eromonas salmonidia; And (ii) inhibiting development into a disease caused by eromonas salmonididae infection.

The term " treatment " or " treatment ", as used herein, refers to (i) inhibition of a disease caused by eromonas salmonidosis; And (ii) alleviating the pathological condition of the disease caused by eromonas salmonididae.

As used herein, the terms "separate", "isolated", or "separated" refer to the separation of bacteriophages from the natural state using various experimental techniques and the securing of features that can identify the bacteriophages of the invention by distinguishing them from other bacteriophages In addition, the present invention also includes propagating the bacteriophage of the present invention industrially so as to utilize it by biotechnology.

The pharmaceutically acceptable carriers to be contained in the composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.

The composition of the present invention contains bacteriophage Aer-SAP-2 as an active ingredient. The bacteriophage Aer-SAP-2 contained therein is contained at a concentration of 1 × 10 ¹ pfu / ml to 1 × 10 ³⁰ pfu / ml or 1 × 10 ¹ pfu / g to 1 × 10 ³⁰ pfu / g, 10 ⁴ pfu / ml to 1 x 10 ¹⁵ pfu / ml or 1 x 10 ⁴ pfu / g to 1 x 10 ¹⁵ pfu / g.

The composition of the present invention may be prepared in a unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. It may be manufactured by inserting it into a multi-capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention may be embodied as a bathing agent and a feed additive, depending on the manner of application, but not limited thereto. Bacteriophages capable of providing an antimicrobial activity against other bacterial species may be added to the composition of the present invention in order to increase efficiency in such a utilization purpose. In addition, other types of bacteriophages having antimicrobial activity against eromonas salmonididae may also be added. Even the bacteriophage having antimicrobial activity against eromonas salmonidida may differ in terms of the strength of antimicrobial activity or the range of antimicrobial activity, so that a proper combination thereof can maximize its effect.

The method for preventing or treating a disease caused by eromonas salmonidia bacteria using the composition comprising the bacteriophage Aer-SAP-2 of the present invention as an active ingredient is more effective than the method based on conventional antibiotics, It is possible to provide an advantage that the specificity to Shidacia is very high. This means that it can be used for the purpose of preventing or treating diseases caused by eromonas salmonidida without affecting other useful microbes, and means that there are very few side effects from the use thereof. Generally, when antibiotics are used, common endophytic bacteria are also harmed, resulting in a decrease in immunity of animals and various side effects due to their use. On the other hand, bacteriophages have a strong antimicrobial activity against bacterial species capable of exhibiting antimicrobial activity even in the case of bacteriophages exhibiting antimicrobial activity. [Bacteriophagus] The range in which the antibacterial activity is exerted. Generally, bacteriophages can exhibit antibacterial activity against some bacterial strains belonging to the same species. In other words, even if belonging to the same bacterium species, there may be differences in susceptibility to bacteriophages depending on the individual bacterium. Therefore, the present invention provides a method for producing a bacteriophage having different antibacterial activity against erythromycin salmonidic acid bacteria Effect can be provided. This provides a big difference in its effectiveness when used in industrial settings.

Figure 1 is an electron micrograph of bacteriophage Aer-SAP-2.

Figure 2 shows the results of experiments showing the ability of bacteriophage Aer-SAP-2 to eromonas salmonidida to kill. Based on the center line of the plate medium, only the buffer containing no bacteriophage Aer-SAP-2 is dispensed on the left side, and the solution containing the bacteriophage Aer-SAP-2 is dispensed on the right side. The transparent part observed on the right is the result of the lysis of bacteria to be tested by the action of bacteriophage Aer-SAP-2.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are merely examples of the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Eromonas You can live.  Isolation of bacteriophage that can kill bacteria

For the isolation of bacteriophages that could kill eromonas salmonididae, samples obtained from natural environment were used. On the other hand, eromonas salmonididae used for bacteriophage isolation was purchased from the environmental microbial bank (prevalence No. KEMB4-337).

More specifically the bacteriophage separation process, erotic Pseudomonas live monitor let a TSB inoculated with the bacteria in the 1 / 1,000 the ratio (T ryptic S oy B roth) medium (Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; Dipeptidium phosphate, 2.5 g / L) were added together and then shake-cultured at 25 DEG C for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. The recovered supernatant was inoculated with eromonas salmonidida at a ratio of 1 / 1,000 and then shake-cultured again at 25 DEG C for 3-4 hours. When the bacteriophage was contained in the sample, this procedure was repeated five times in total so that the number of bacteriophages could be sufficiently increased. After 5 replicates, the culture was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. Through a conventional spot assay using the thus obtained filtrate, the presence of bacteriophage capable of killing eromonas salmonidia was examined.

The above drop test was carried out as follows. The TSB medium was inoculated with eromonas salmonidida at a ratio of 1 / 1,000, and then cultured with shaking at 25 DEG C overnight. The thus prepared 3 ㎖ culture solution of the erosion Pseudomonas live monitor let bacteria (OD ₆₀₀ is 1.5) TSA (T ryptic S oy A gar) plate medium (Casein Digest, 15 g / L; Soy bean digest, 5 g / L; NaCl , 5 g / L; agar, 15 g / L). The smear medium was allowed to stand in a clean bench for about 30 minutes to allow the smear solution to dry. After drying, 10 μl of the filtrate prepared above was spotted onto a flat plate medium on which eromonas salmonidase was plated. This was allowed to stand for 30 minutes and then dried. After drying, the plate culture medium was incubated at 25 ° C. for one day. Then, a clear zone was formed at the position where the filtrate was separated. In the case of the filtrate in which the transparent ring is formed, it can be judged that bacteriophage capable of killing eromonas salmonidida is included. Through these investigations, it was possible to obtain a filtrate containing bacteriophage with the ability to kill eromonas salmonididae.

Pure bacteriophage was isolated by using the filtrate which showed the existence of bacteriophage having killing ability against eromonas salmonidida. For the separation of pure bacteriophage, a usual plaque assay was used. To explain this in detail, one of the broths formed in the leavening assay was recovered using a sterilized tip, and then added to the culture solution of eromonas salmonidis, followed by incubation at 25 ° C. for 4-5 hours. After incubation, supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. The culture medium of eromonas salmonidicum was added to the obtained supernatant in a volume of one-fifth of the volume, followed by further incubation at 25 ° C for 4-5 hours. This procedure was performed at least 5 times to increase the number of bacteriophages, and finally the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. The obtained supernatant was used for the analysis of the washing solution. Since the separation of the pure bacteriophage is not normally achieved by only one step of the above procedure, the former step is repeated again by using the formed agitation blank. This procedure was repeated at least five times to obtain a solution containing pure bacteriophage. The separation of pure bacteriophages was usually repeated until the size and shape of the formed lysate were all similar. Finally, it was confirmed by electron microscopic analysis whether the bacteriophage was purely isolated. The procedure described above was repeated until pure isolation was confirmed by electron microscopy. Electron microscopic analysis was carried out according to a conventional method. A brief explanation is as follows. The solution containing the pure bacteriophage was embedded in a copper grid, followed by negative staining and drying with 2% Uranyl acetate, followed by observation through a transmission electron microscope. An electron micrograph of the purely isolated bacteriophage is shown in FIG. Judging from the morphological characteristics, it was confirmed that the newly acquired bacteriophage belongs to the Myoviridae bacteriophage.

The solution containing pure bacteriophage identified in this way was subjected to the following purification procedure. To the solution containing the pure bacteriophage, the culture of eromonas salmonidicum was added in a volume of one-half of the total volume of the solution, followed by further incubation for 4-5 hours. After incubation, supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This process was repeated five times in total to obtain a solution containing a sufficient number of bacteriophages. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter, and then a conventional polyethylene glycol (PEG) precipitation process was performed. Specifically, PEG and NaCl were added to 100 ml of the filtrate to make 10% PEG 8000 / 0.5 M NaCl, and the mixture was allowed to stand at 4 ° C for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate . Thus obtained precipitate bacteriophage buffer (Buffer; 10 mM Tris-HCl , 10 mM MgSO 4, 0.1% Gelatin, pH 8.0) and suspended in 5 ㎖. This is called a bacteriophage suspension or bacteriophage solution.

The purified bacteriophage was named as Bacteriophage Aer-SAP-2 and deposited on September 22, 2015 with the BRC (Korea Research Institute of Bioscience and Biotechnology) (Accession No. KCTC 12909BP ).

Example  2: Bacteriophage Aer - SAP -2 genome sequencing and genome sequencing

The genome of bacteriophage Aer-SAP-2 was isolated as follows. For the dielectric separation, the bacteriophage suspension obtained by the same method as in Example 1 was used. In order to remove the DNA and RNA of eromonas salmonidase, which may be contained in the supernatant, 200 U of DNase I and 200 A of RNase A were added to 10 ml of the bacteriophage suspension, and the mixture was left at 37 ° C for 30 minutes. After 30 minutes of incubation, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added to remove DNase I and RNase A activity, and the mixture was allowed to stand for another 10 minutes. After incubation for another 10 min at 65 ° C, 100 μl of proteinase K (20 mg / ml) was added to dissociate the bacteriophage outer wall, followed by reaction at 37 ° C for 20 minutes. Then, 500 μl of 10% sodium dodecyl sulfate (SDS) was added, and then reacted again at 65 ° C for 1 hour. After 1 hour of reaction, 10 ml of a mixture of phenol: chloroform: isoamylalcohol having a composition ratio of 25: 24: 1 was added to the reaction solution, and the mixture was mixed well. Then, the mixture was centrifuged at 13,000 rpm for 15 minutes to separate the layers. The upper layer was taken out of the separated layers, 1.5 parts by volume of isopropyl alcohol was added thereto, and the mixture was centrifuged at 13,000 rpm for 10 minutes The dielectric was precipitated. After the precipitate was collected, 70% ethanol was added to the precipitate, and the precipitate was further washed by centrifugation at 13,000 rpm for 10 minutes. The washed precipitate was recovered, vacuum dried and dissolved in 100 μl of water. The above procedure was repeated to secure a large amount of the genome of bacteriophage Aer-SAP-2.

The genome thus obtained was subjected to next generation sequencing analysis using the illumina Mi-Seq instrument in Macrogen, and the genome sequence information of bacteriophage Aer-SAP-2 was obtained. The finally analyzed bacteriophage Aer-SAP-2 genome has a size of 53,870 bp and the entire genomic sequence is shown in SEQ ID NO: 1.

Based on the genomic sequence information of the obtained bacteriophage Aer-SAP-2, similarity to previously known bacteriophage genomic sequences was examined using BLAST on the web. As a result of BLAST analysis, bacteriophage sequences having homology of more than 50% were not confirmed.

Based on this fact, it was concluded that bacteriophage Aer-SAP-2 is a new bacteriophage different from the previously reported bacteriophages. In addition to these facts, it is believed that bacteriophage Aer-SAP-2 can provide different antimicrobial effects from other bacteriophages reported from the fact that the different kinds of bacteriophages usually provide different antimicrobial power and antimicrobial range there was.

Example  3: Bacteriophage Aer - SAP -2 of Eromonas You can live.  For bacteria Destructiveness  Research

The ability of the isolated bacteriophage Aer-SAP-2 to eromonas salmonidida was investigated. The extinction ability was investigated by examining whether or not a transparent ring was formed through the drip test as described in Example 1. The Eromonas salmonidida strains used for the study of the killing activity were 17 weeks in total, which were either purchased through the strain bank or isolated by the present inventors and identified as Eromonas salmonididae. Bacteriophage Aer-SAP-2 has an ability to kill for 15 weeks in total, including KEMB4-337 strain (KEMB: Korea Environmental Microorganisms Bank, Environmental Microbial Bank), among the 17 weeks of eromonas salmonidia there was. Representative experimental results are shown in Fig. On the other hand, Edwardsiella tarda of Bacteriophage Aer-SAP-2, Vibrio anguillarum , Vibrio ichthyoenteri , Lactococcus Garvieae , Streptococcus parauberis , Streptococcus iniae , and Aeromonas spp. hydrophila ). As a result, bacteriophage Aer-SAP-2 did not have the ability to kill bacteria of these species.

As a result, it was confirmed that the bacteriophage Aer-SAP-2 has excellent killing ability against eromonas salmonidida and can exert antibacterial effect against many eromonas salmonidia strains. This means that bacteriophage Aer-SAP-2 can be used as an active ingredient of a composition for the prevention or treatment of diseases caused by eromonas salmonidosis.

Example  4: Bacteriophage Aer - SAP -2 of Eromonas You can live.  For prevention of fungal infection Experimental Example

Add 100 μl of bacteriophage Aer-SAP-2 at a level of 1 × 10 ⁸ pfu / ml to one tube containing 9 ml of TSB medium, and add the same volume of TSB medium to the tube containing 9 ml of TSB medium. Lt; / RTI > Then, the culture solution of Eromonas salmonidicum was added to each tube so that the absorbance was about 0.5 at 600 nm. After the addition of eromonas salmonididae, the tubes were transferred to an incubator at 25 ° C. and cultured under shaking to observe the growth state of eromonas salmonididae. As shown in Table 1, growth inhibition of eromonas salmonidia was observed in the tubes added with the bacteriophage Aer-SAP-2 solution, whereas in the tubes without the bacteriophage solution, the growth of eromonas salmonidia No inhibition was observed.

Growth inhibition of eromonas salmonididae

division	Absorbance value
	Culture 0 min	60 minutes after incubation	120 minutes after incubation
Bacteriophage fluid addition	0.48	1.12	1.67
Bacteriophage solution addition	0.48	0.23	0.14

From these results, it was confirmed that the bacteriophage Aer-SAP-2 of the present invention not only inhibits the growth of eromonas salmonidicum but also has the ability to kill eromonas salmonidosis. From this, it is confirmed that bacteriophage Aer-SAP- -2 could be used as an effective ingredient of a composition for the prevention of diseases caused by eromonas salmonididae.

Example  5: Bacteriophage Aer - SAP -2 Eromonas You can live.  Bacterium-induced Preventive Animal Test for Disease

Rainbow trout (average weight: 23.4 g, average length: 15.8 cm) was divided into two groups of 20 rats and separated for 14 days in a water tank. The ambient environment of the water tank was controlled, and the temperature of the laboratory with the water tank was kept constant. Feeds containing 1 × 10 ⁸ pfu / g of bacteriophage Aer-SAP-2 were fed to rainbow trout in the experimental group (bacteriophage-treated group) from the start of the experiment to the experimental period according to a conventional feed feeding method. On the other hand, the rainbow trout of the control group (bacteriophage MIT) received feeds of the same composition without bacteriophage Aer-SAP-2 in the same manner. From the 7th day after the start of the test, eromonas salmonididae were added to feeds at a level of 1 × 10 ⁸ cfu / g for 2 days, and fed twice a day to induce eromonas salmonidia infection . On the 9th day after the start of the test, the incidence of the incidence of pests was examined in all test animals on a daily basis. The incidence of sprouting was measured by measuring the ulcer size of the body surface. Ulcer size (US) score (normal (no ulcer): 0, weak ulcer (ulcer size: less than 0.5 cm): 1, strong ulcer (ulcer size: Were measured. The results are shown in Table 2.

Body size ulcer size measurement (average)

	US score (mean)
date	D9	D10	D11	D12	D13	D14
Control group (female bacteriophage)	0.40	0.45	0.60	0.65	0.75	0.80
Experimental group (bacteriophage administration)	0.10	0.05	0	0	0	0

From these results, it was confirmed that the bacteriophage Aer-SAP-2 of the present invention is highly effective in the prevention of diseases caused by eromonas salmonidosis.

Example  6: Bacteriophage Aer - SAP -2 Eromonas You can live.  For diseases caused by bacteria Treatment example

The therapeutic effect of bacteriophage Aer-SAP-2 on diseases caused by eromonas salmonididae was investigated. 40 rabbits (average weight: 23.6 g, average length: 15.8 cm) were divided into two groups and divided into two groups. The ambient environment of the water tank was controlled, and the temperature of the laboratory with the water tank was kept constant. Feeds contaminated with eromonas salmonidia were fed twice a day in a conventional feed feeding manner at a level of 1 × 10 ⁸ cfu / g for 3 days from the 5th day after the start of the experiment. From the last day of contaminated feed, eromonas salmonididae were identified in both tanks with clinical symptoms. The rainbow trout of the experimental group (bacteriophage-treated group) included bacteriophage Aer-SAP-2 from the day after the contaminated feeding of eromonas salmonidida for 3 days (the 8th day after the start of the test) ⁸ pfu / g) were fed according to the conventional feed feeding method. On the other hand, the rainbow trout of the control group (bacteriophage MIT) received feeds of the same composition without bacteriophage Aer-SAP-2 in the same manner. From the third day after the forced infection of eromonas salmonididae (on the eighth day of the test), the incidence of infestation was examined in all test animals on a daily basis. The incidence of incontinence caused by eromonas salmonidida was measured by measuring the ulcer size of the body surface as in Example 5. The results are shown in Table 3.

Body size ulcer size measurement (average)

	US score (mean)
date	D8	D9	D10	D11	D12	D13	D14
Control group (female bacteriophage)	0.95	1.20	1.45	1.50	1.60	1.55	1.60
Experimental group (bacteriophage administration)	0.95	0.85	0.40	0.30	0.20	0.15	0.10

From these results, it was confirmed that the bacteriophage Aer-SAP-2 of the present invention is very effective for the treatment of diseases caused by eromonas salmonidosis.

Example  7: Preparation of feed additive and feed

The feed additive was prepared so that the bacteriophage Aer-SAP-2 contained 1 x 10 ⁸ pfu of bacteriophage Aer-SAP-2 per gram of feed additive. The feed additives were prepared by adding maltodextrin to the bacteriophage solution (50%, w / v) followed by lyophilization. And finally pulverized into a fine powder form. The drying process during the manufacturing process may be replaced by vacuum drying, warm drying, or drying at room temperature. For the control experiment, a feed additive without bacteriophage was also prepared by using the buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0) used in the preparation of the bacteriophage solution instead of the bacteriophage .

Each of the two feed additives was mixed with a 250 - fold amount of raw fish to produce two final feeds.

Example  8: Bath  Produce

The bath preparation was prepared as follows. Bacteriophage Aer-SAP-2 solution was used to prepare a bathing agent so that 1 × 10 ⁸ pfu of bacteriophage Aer-SAP-2 per 1 ml of bath was contained. The manufacturing method of the bathing agent is such that the bacteriophage Aer-SAP-2 solution is added so as to contain 1 × 10 ⁸ pfu of bacteriophage Aer-SAP-2 per 1 ml of the buffer used for producing the bacteriophage solution, Respectively. For the control experiment, the buffer solution used in the preparation of the bacteriophage solution was used as the non-bacteriophage-free bath solution.

The two bath preparations thus prepared were diluted with water at a volume ratio of 1,000 times and used as a final bathing agent.

Example  9: Confirmation of the effect of specification on rainbow trout breeding

Using the feeds prepared in Example 7 and Example 8, and a bathing agent, the improvement of the specification results in rainbow trout breeding was investigated. In particular, the survey was conducted in terms of mortality. A total of 200 rainbow trout were divided into two groups of 100 rats divided into two groups (feed-fed group-A, bath-treated group-B) for four weeks. Each group was divided into small groups consisting of 50 animals. Each subgroup was divided into small group (small group-1) with bacteriophage Aer-SAP-2 and small group (small group -②) without bacteriophage. The rainbow trout, which was subject to the test, was a 5 - week - old rainbow trout (average weight: 23.8 g, average length: 15.9 cm). Rainbow trout of each test group were kept in separate tanks at regular intervals. Each subgroup is identified and named as shown in Table 4 below.

Subgroup classification and display in the rainbow trout specimen test

apply	Classification and display of small groups
	Application of bacteriophage Aer-SAP-2	Bacteriophage not applied
Group fed with feed	A-1	A-2
A group treated with bath	B-1	B-2

In the case of feed feeding, the feed prepared in Example 7 was fed according to a conventional feed feeding method according to the classification of Table 4, and in the case of the bath treatment, the feed prepared in accordance with the preparation method of bath preparation described in Example 8 A bath agent was treated according to a conventional bath bath treatment method in which a fish body was immersed in a diluting solution of a bath agent according to Table 4. The results are shown in Table 5.

Mortality in rainbow trout specimens

group	Number of dead / test population	Mortality (%)
A-1	3/50	6.0
A-2	11/50	22.0
B-1	4/50	8.0
B-2	15/50	30.0

As a result, it was confirmed that the feeding of the feed prepared according to the present invention and the treatment of the bath agent according to the present invention were effective in reducing the mortality in rainbow trout breeding. From this, it can be concluded that the application of the composition of the present invention is effective in improving the specification results of rainbow trout.

It will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

[Access number]

Depositor Name: KCTC

Accession number: KCTC 12909BP

Funding date: 20150922

Claims (7)

1. The mio viridis versus bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) isolated from nature, having the ability to kill eromonas salmonididae and having the genome of SEQ ID NO: 1.
2. A composition for preventing or treating diseases caused by eromonas salmonididae comprising the bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) of claim 1 as an active ingredient.
3. The composition according to claim 2, wherein the composition is used in the form of a bathing agent or a feed additive, wherein the composition is for preventing or treating diseases caused by eromonas salmonidia.
4. Which comprises administering to a composition comprising the bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) according to claim 2 as an active ingredient, an immobilization of an animal other than a human. A method for preventing or treating a disease.
5. 5. The method according to claim 4, wherein the composition is in the form of a bath medicine.
6. A method for preventing or preventing a disease caused by eromonas salmonidia bacteria, which comprises the step of administering a composition comprising the bacteriophage Aer-SAP-2 (Accession No. KCTC 12909BP) according to claim 2 as an active ingredient to an animal other than man. Or treatment.
7. 7. The method according to claim 6, wherein the composition is in the form of a feed additive.

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